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Results and Future Plans

Functional Genomics Research Group
Developing Mouse Models for Human Diseases and Enhancing Their Utility as a Resource

Toshihiko Shiroishi

Professor, Mammalian Genetics Laboratory, National Institute of Genetics

Professor Shiroishi served primarily as a principal investigator directing research at the Mammalian Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics (which is a core institution of the National BioResource Project). His group conducted mouse ENU mutagenesis at GSC.

Over the past ten years, research directions of the genomic sciences have shifted "from genome sequencing to functional genomics". To provide a strong methodological basis for functional genomic studies, new strategies have been created whereby a vast number of mutant animals with functionally abnormal genes are generated and subjected to detailed analysis for their phenotypic expression. About ten years ago, large-scale projects devoted to this purpose were initiated in many countries. At GSC, large-scale "mouse mutagenesis projects" were officially started in 1999, and are based on the development of mouse models using the chemical mutagen N-ethyl-N-nitrosourea (ENU). In addition, multiple mutant mouse strains have been developed as an animal model for studying human diseases and, in particular, promoting understanding of the functions of genes. In 2002, our group joined the "National BioResource Project" (which covers outsourced research programs under the Ministry of Education, Culture, Sports, Science and Technology) which for 5 years had been engaged in the development of animal models of human lifestyle-related diseases and dementia, among others. An outline of our group's research progress follows.

Identifying candidate mouse models of human disease

As a basis for advancing our understanding of the pathology of human diseases, a systematic and comprehensive phenotype analysis platform which includes about 400 subtests has been established. Over 13,000 mice were screened for dominant and recessive mutations. So far, over 400 mutant strains of candidate models of human diseases have been developed. Among these, 348 strains were deposited in the RIKEN BioResource Center. In addition, for identification of causative genes, a high-throughput gene mapping system using SNP markers has been developed. The chromosomal loci of causative genes in 248 strains have been determined.
Furthermore, using an "inference system for causative genes of human disease" (developed in collaboration with the Omics Bioinformatics Team), we assessed the efficiency of causative gene identification. The causative genes for 62 strains have thus been determined.

Development of viable mouse models

Over 340 strains of mutant mice have been deposited in the database

The mouse shown on the left was generated as a mutant for type II diabetes (the mouse shown on the right is a littermate with normal phenotype). Up to March 2007, the phenotypes of 348 mutant strains were characterized and deposited in our mutant mouse database. The sperms of about 10,000 individual mice are stored in liquid nitrogen, which is also the base for the RIKEN gene-driven mutagenesis. Established mutant strains are available to external users via requests to the RIKEN BioResource Center (www.brc.riken.jp/lab/animal/en/gscmouse.shtml).

Using many mutant strains generated as candidate models of human diseases, we conducted detailed and comprehensive phenotypic characterization and identification/functional studies of causative genes in the pathogenesis of diseases. Over 50 mutant strains have been developed for studies of human lifestyle-related diseases including models of diabetes, hyperlipidemia, cancer, high blood pressure, and osteoporosis.
Besides human lifestyle-related diseases, models with potential applications for behavioral disorders, auditory disturbance, and retinal dysfunction have been successfully developed. Among 70 mutant strains, causative genes and mutations have been identified for over 50% of these models, which include novel genes implicated in the pathogenesis of relevant diseases. These results will contribute to understanding the development of symptoms mechanisms of human diseases.

Establishing rapid mutation discovery systems

We have developed a high-throughput mutation discovery system capable of screening point mutations*1 of target genes in mutant strains, with as many as 10,000 strains of ENU mutant mice (mutant mouse library) being included in the studies. Through this strategy the number of target genes available for screening expanded to 251 in the year 2006. More than 70% of these genes were screened upon request by external researchers. More than 400 mutations have been identified, for which various sequence alterations have been classified: amino acid substitutions (61%), nonsense mutations/splicing variants (10%), and synonymous substitutions (29%).
The identified mutations have been reconstituted in more than 70 strains of mice to date. Functional studies of genes were carried out in collaboration with external researchers, and examples of established models include those of schizophrenia, depression and hyperdactylia.
Based on such progress, we hope to continue and undertake studies in collaboration with the RIKEN BioResource Center to develop the "bioresource infrastructure" and to support a wide spectrum of research in the life sciences in the following 4 areas.

*1 Point mutations
Mutations involving the replace-ment of a single nucleotide with another in the DNA sequence, which may arise from DNA replication errors, mutagen treatment, UV irradiation, etc.

1. Platform for the Japanese Mouse Clinic

We have re-constructed a platform for the Japanese Mouse Clinic, which features a hierarchical system of mouse phenotype characterization derived from a previous platform of phenotype analysis for ENU mutagenesis. Using this new system, we carry out phenotype characterization of mutant mouse strains (created by ENU-induced mutagenesis, gene knockouts, etc.) deposited mainly at the RIKEN BioResource Center, focusing on maximizing the utility of bioresources in the form of phenotype data.

2. Development of phenotypic characterization systems

In order to enhance the values of the many mouse mutants at the RIKEN BioResource Center as human diseases models, the high-sensitivity systems for phenotype characterization have been developed using advanced methods in proteomics and in vivo imaging.
Furthermore, we will collaborate with other groups in the research community and leading clinical institutions to develop and study some of the representative model systems available at the RIKEN BioResource Center. With this infrastructure, our group works on translational research based on the development of useful models for studying human diseases as well as identification of novel genes associated with human diseases.

3. Gene-driven mutagenesis and the mutant library

The RIKEN mutant mouse library, which was independently developed in Japan, is a resource for reverse genetics studies, with a large reservoir of mutant strains representing 180,000 amino acid substitutios and 30,000 gene knockouts. This library was started ahead of similar large-scale projects undertaken in Europe and the US, and its scale is being further expanded through partnership with external researchers in the community. To maintain our independent and leading position, we are working to improve our capacity by, for example, increasing the size of the library, enhancing the mutation discovery system, and developing a primary assessment system for molecular phenotyping.

4. Facilitating global access to phenotype data

To enable world-scale integration of mouse phenotype data, it becomes an issue of common interest to the international research community that standardization of terminology*2 with informational technology such as ontology, and to share information generated through their phenotypic characterization platforms (data production infrastructure). In the future, we will develop a globally accessible mouse phenotype database that enables systematic retrieval of phenotype information with the international standardized evaluation to deposit mouse resources available at the RIKEN BioResource Center, which make up 20% of the world total. This activity may greatly contribute to the development of the international knowledge infrastructures in life science.

*2 Ontology technologies
Ontology literally means the study of being or existence. Here, it refers to technologies associated with new terminology, which are used for specific concepts and contexts.

ENU mutagenesis project

This project involves the generation of mutant strains through random mutations in the wild-type mouse genome, and functional studies of the identified mutations of interest coupled to phenotypes.

Functional Network

Development of an inferential prediction system (PosMed) based on phenotype data for genes identified with transforming mutations.